Regulation of iron homeostasis is essential for the virulence of the opportunistic fungal pathogen Aspergillus fumigatus.1 The cytosolic monothiol glutaredoxin GrxD was recently shown to play a critical role in iron metabolism via regulation of iron-sulfur (Fe-S) binding iron-responsive transcription factors and interaction with components of the cytosolic Fe-S cluster biogenesis pathway.2 Interestingly, the putative copper-binding metallothionein CmtA was also identified as a binding partner for GrxD; however, the metal binding properties of both proteins and the nature of their interactions were unclear. Here, we addressed these open questions by recombinant expression and purification of AfGrxD and AfCmtA and analysis of their metal-protein and protein-protein interactions. Using elemental analysis and UV-visible absorption, CD, EPR, EXAFS, and Mössbauer spectroscopies, we demonstrate that AfCmtA expressed in E. coli primarily binds a [2Fe-2S]2+ cluster, mononuclear Fe2+, and Zn2+, while AfGrxD ligates [2Fe-2S]2+ and [3Fe-4S]+ clusters at its dimer interface. Furthermore, we discovered that the interaction between AfGrxD and AfCmtA involves unidirectional transfer of a [2Fe-2S]2+ cluster from AfGrxD to AfCmtA. Accordingly, co-expression of AfGrxD with AfCmtA in E. coli increased in vivo Fe-S cluster loading in AfCmtA, supporting a role for AfGrxD in the delivery of [2Fe-2S]+2 clusters to AfCmtA. Taken together, these results suggest that the sole metallothionein in Aspergillus fumigatus represents a unique class of fungal metallothioneins that may primarily store and/or deliver Fe-S clusters and Zn2+.